B‐cell precursor acute lymphoblastic leukemia (BCP‐ALL) is a genetically heterogeneous blood cancer characterized by abnormal expansion of immature B cells. Although intensive chemotherapy provides ...high cure rates in a majority of patients, subtypes harboring certain genetic lesions, such as MLL rearrangements or BCR‐ABL1 fusion, remain clinically challenging, necessitating a search for other therapeutic approaches. Herein, we aimed to validate antioxidant enzymes of the thioredoxin system as potential therapeutic targets in BCP‐ALL. We observed oxidative stress along with aberrant expression of the enzymes associated with the activity of thioredoxin antioxidant system in BCP‐ALL cells. Moreover, we found that auranofin and adenanthin, inhibitors of the thioredoxin system antioxidant enzymes, effectively kill BCP‐ALL cell lines and pediatric and adult BCP‐ALL primary cells, including primary cells cocultured with bone marrow‐derived stem cells. Furthermore, auranofin delayed the progression of leukemia in MLL‐rearranged patient‐derived xenograft model and prolonged the survival of leukemic NSG mice. Our results unveil the thioredoxin system as a novel target for BCP‐ALL therapy, and indicate that further studies assessing the anticancer efficacy of combinations of thioredoxin system inhibitors with conventional anti‐BCP‐ALL drugs should be continued.
In B‐cell precursor acute lymphoblastic leukemia (BCP‐ALL), we observed oxidative stress along with increased expression of the enzymes of the thioredoxin antioxidant system. Inhibition of these enzymes with auranofin and adenanthin triggered oxidative and endoplasmic reticulum stress and effectively killed BCP‐ALL cells in vitro and in vivo. Our results unveil thioredoxin system as a novel target for BCP‐ALL therapy.
L-ascorbate (L-ASC) is a widely-known dietary nutrient which holds promising potential in cancer therapy when given parenterally at high doses. The anticancer effects of L-ASC involve its ...autoxidation and generation of H
2
O
2
, which is selectively toxic to malignant cells. Here we present that thioredoxin antioxidant system plays a key role in the scavenging of extracellularly-generated H
2
O
2
in malignant B-cells. We show that inhibition of peroxiredoxin 1, the enzyme that removes H
2
O
2
in a thioredoxin system-dependent manner, increases the sensitivity of malignant B-cells to L-ASC. Moreover, we demonstrate that auranofin (AUR), the inhibitor of the thioredoxin system that is used as an antirheumatic drug, diminishes the H
2
O
2
-scavenging capacity of malignant B-cells and potentiates pharmacological ascorbate anticancer activity
in vitro
and
in vivo
. The addition of AUR to L-ASC-treated cells triggers the accumulation of H
2
O
2
in the cells, which results in iron-dependent cytotoxicity. Importantly, the synergistic effects are observed at as low as 200 µM L-ASC concentrations. In conclusion, we observed strong, synergistic, cancer-selective interaction between L-ASC and auranofin. Since both of these agents are available in clinical practice, our findings support further investigations of the efficacy of pharmacological ascorbate in combination with auranofin in preclinical and clinical settings.
fx1
•
Lack of peroxiredoxin 1 potentiates antileukemic activity of L-ascorbate
in vitro
and
in vivo.
•
Auranofin and L-ascorbate synergistically kill malignant B cells.
•
Auranofin leads to intracellular accumulation of H
2
O
2
generated by L-ascorbate.
•
Auranofin and L-ascorbate trigger iron-dependent oxidative damage and cytotoxicity.
The prognosis for B-cell precursor acute lymphoblastic leukemia patients with Mixed-Lineage Leukemia (MLL) gene rearrangements (MLLr BCP-ALL) is still extremely poor. Inhibition of anti-apoptotic ...protein BCL-2 with venetoclax emerged as a promising strategy for this subtype of BCP-ALL, however, lack of sufficient responses in preclinical models and the possibility of developing resistance exclude using venetoclax as monotherapy. Herein, we aimed to uncover potential mechanisms responsible for limited venetoclax activity in MLLr BCP-ALL and to identify drugs that could be used in combination therapy. Using RNA-seq, we observed that long-term exposure to venetoclax in vivo in a patient-derived xenograft model leads to downregulation of several tumor protein 53 (TP53)-related genes. Interestingly, auranofin, a thioredoxin reductase inhibitor, sensitized MLLr BCP-ALL to venetoclax in various in vitro and in vivo models, independently of the p53 pathway functionality. Synergistic activity of these drugs resulted from auranofin-mediated upregulation of NOXA pro-apoptotic protein and potent induction of apoptotic cell death. More specifically, we observed that auranofin orchestrates upregulation of the NOXA-encoding gene Phorbol-12-Myristate-13-Acetate-Induced Protein 1 (PMAIP1) associated with chromatin remodeling and increased transcriptional accessibility. Altogether, these results present an efficacious drug combination that could be considered for the treatment of MLLr BCP-ALL patients, including those with TP53 mutations.
L-ascorbate (L-ASC) is a widely-known dietary nutrient which holds promising potential in cancer therapy when given parenterally at high doses. The anticancer effects of L-ASC involve its ...autoxidation and generation of H
O
, which is selectively toxic to malignant cells. Here we present that thioredoxin antioxidant system plays a key role in the scavenging of extracellularly-generated H
O
in malignant B-cells. We show that inhibition of peroxiredoxin 1, the enzyme that removes H
O
in a thioredoxin system-dependent manner, increases the sensitivity of malignant B-cells to L-ASC. Moreover, we demonstrate that auranofin (AUR), the inhibitor of the thioredoxin system that is used as an antirheumatic drug, diminishes the H
O
-scavenging capacity of malignant B-cells and potentiates pharmacological ascorbate anticancer activity in vitro and in vivo. The addition of AUR to L-ASC-treated cells triggers the accumulation of H
O
in the cells, which results in iron-dependent cytotoxicity. Importantly, the synergistic effects are observed at as low as 200 µM L-ASC concentrations. In conclusion, we observed strong, synergistic, cancer-selective interaction between L-ASC and auranofin. Since both of these agents are available in clinical practice, our findings support further investigations of the efficacy of pharmacological ascorbate in combination with auranofin in preclinical and clinical settings.
Liquid biopsy based on cell-free DNA circulating in plasma has shown solid results as a non-invasive biomarker. In the present study we evaluated the utility of circulating free DNA (cfDNA) and the ...sub-type tumor DNA (ctDNA) in hepatocellular cancer (HCC) patients to assess therapy response and clinical outcome.
A cohort of 13 patients recruited in the context of the SORAMIC trial with unresectable, advanced HCC and different etiological and clinicopathological characteristics was included in this exploratory study. Plasma samples were collected between liver micro-intervention and beginning of sorafenib-based systemic therapy and then in correspondence of three additional follow-ups. DNA was isolated from plasma and next generation sequencing (NGS) was performed on a panel of 597 selected cancer-relevant genes.
cfDNA levels showed a significant correlation with the presence of metastases and survival. In addition cfDNA kinetic over time revealed a trend with the clinical history of the patients, supporting its use as a biomarker to monitor therapy. NGS-based analysis on ctDNA identified 28 variants, detectable in different combinations at the different time points. Among the variants, HNF1A, BAX and CYP2B6 genes showed the highest mutation frequency and a significant association with the patients' clinicopathological characteristics, suggesting a possible role as driver genes in this specific clinical setting.
Taken together, the results support the prognostic value of cfDNA/ctDNA in advanced HCC patients with the potential to predict therapy response. These findings support the clinical utility of liquid biopsy in advanced HCC improving individualized therapy and possible earlier identification of treatment responders.
The underlying etiology of ischemic stroke remains unknown in up to 30% of patients.
This study explored the causal role of complicated (American Heart Association-lesion type VI) nonstenosing ...carotid artery plaques (CAPs) in cryptogenic stroke (CS).
CAPIAS (Carotid Plaque Imaging in Acute Stroke) is an observational multicenter study that prospectively recruited patients aged older than 49 years with acute ischemic stroke that was restricted to the territory of a single carotid artery on brain magnetic resonance imaging (MRI) and unilateral or bilateral CAP (≥2 mm, NASCET North American Symptomatic Carotid Endarterectomy Trial <70%). CAP characteristics were determined qualitatively and quantitatively by high-resolution, contrast-enhanced carotid MRI at 3T using dedicated surface coils. The pre-specified study hypotheses were that that the prevalence of complicated CAP would be higher ipsilateral to the infarct than contralateral to the infarct in CS and higher in CS compared with patients with cardioembolic or small vessel stroke (CES/SVS) as a combined reference group. Patients with large artery stroke (LAS) and NASCET 50% to 69% stenosis served as an additional comparison group.
Among 234 recruited patients, 196 had either CS (n = 104), CES/SVS (n = 79), or LAS (n = 19) and complete carotid MRI data. The prevalence of complicated CAP in patients with CS was significantly higher ipsilateral (31%) to the infarct compared with contralateral to the infarct (12%; p = 0.0005). Moreover, the prevalence of ipsilateral complicated CAP was significantly higher in CS (31%) compared with CES/SVS (15%; p = 0.02) and lower in CS compared with LAS (68%; p = 0.003). Lipid-rich and/or necrotic cores in ipsilateral CAP were significantly larger in CS compared with CES/SVS (p < 0.05).
These findings substantiate the role of complicated nonstenosing CAP as an under-recognized cause of stroke. (Carotid Plaque Imaging in Acute Stroke CAPIAS; NCT01284933).
Complicated nonstenosing carotid artery plaques (CAPs) are an under-recognized cause of stroke.
The purpose of this study was to determine whether complicated CAP ipsilateral to acute ischemic ...anterior circulation stroke (icCAP) are associated with recurrent ischemic stroke or transient ischemic attack (TIA).
The CAPIAS (Carotid Plaque Imaging in Acute Stroke) multicenter study prospectively recruited patients with ischemic stroke restricted to the territory of a single carotid artery. Complicated (AHA-lesion type VI) CAP were defined by multisequence, contrast-enhanced carotid magnetic resonance imaging obtained within 10 days from stroke onset. Recurrent events were assessed after 3, 12, 24, and 36 months. The primary outcome was recurrent ischemic stroke or TIA.
Among 196 patients enrolled, 104 patients had cryptogenic stroke and nonstenosing CAP. During a mean follow-up of 30 months, recurrent ischemic stroke or TIA occurred in 21 patients. Recurrent events were significantly more frequent in patients with icCAP than in patients without icCAP, both in the overall cohort (incidence rate 3-year interval: 9.50 vs 3.61 per 100 patient-years; P = 0.025, log-rank test) and in patients with cryptogenic stroke (10.92 vs 1.82 per 100 patient-years; P = 0.003). The results were driven by ipsilateral events. A ruptured fibrous cap (HR: 4.91; 95% CI: 1.31-18.45; P = 0.018) and intraplaque hemorrhage (HR: 4.37; 95% CI: 1.20-15.97; P = 0.026) were associated with a significantly increased risk of recurrent events in patients with cryptogenic stroke.
Complicated CAP ipsilateral to acute ischemic anterior circulation stroke are associated with an increased risk of recurrent ischemic stroke or TIA. Carotid plaque imaging identifies high-risk patients who might be suited for inclusion into future secondary prevention trials. (Carotid Plaque Imaging in Acute Stroke CAPIAS; NCT01284933)
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The development of two conventional dendritic cells (DC) subsets (cDC1 and cDC2) and the plasmacytoid DC (pDC) in vivo and in cultures of bone marrow (BM) cells is mediated by the growth factor ...Flt3L. However, little is known about the factors that direct the development of the individual DC subsets. Here, we describe the selective in vitro generation of murine ESAMlow CD103− XCR1− CD172a+ CD11b+ cDC2 from BM by treatment with a combination of Flt3L, LIF, and IL‐10 (collectively named as FL10). FL10 promotes common dendritic cell progenitors (CDP) proliferation in the cultures, similar to Flt3L and CDP sorted and cultured in FL10 generate exclusively cDC2. These cDC2 express the transcription factors Irf4, Klf4, and Notch2, and their growth is reduced using BM from Irf4−/− mice, but the expression of Batf3 and Tcf4 is low. Functionally they respond to TLR3, TLR4, and TLR9 signals by upregulation of the surface maturation markers MHC II, CD80, CD86, and CD40, while they poorly secrete proinflammatory cytokines. Peptide presentation to TCR transgenic OT‐II cells induced proliferation and IFN‐γ production that was similar to GM‐CSF‐generated BM‐DC and higher than Flt3L‐generated DC. Together, our data support that FL10 culture of BM cells selectively promotes CDP‐derived ESAMlow cDC2 (cDC2B) development and survival in vitro.
In vitro culture of murine bone marrow cells in Flt3L + LIF + IL‐10 promotes CD11b+ ESAMlow cDC2B subset generation from CDP but blocks cDC1 and pDC development. Flt3L alone or combined does not induce cDC2A generation in vitro.
The development of two conventional dendritic cells (DC) subsets (cDC1 and cDC2) and the plasmacytoid DC (pDC) in vivo and in cultures of bone marrow (BM) cells is mediated by the growth factor ...Flt3L. However, little is known about the factors that direct the development of the individual DC subsets. Here, we describe the selective in vitro generation of murine ESAM
CD103
XCR1
CD172a
CD11b
cDC2 from BM by treatment with a combination of Flt3L, LIF, and IL-10 (collectively named as FL10). FL10 promotes common dendritic cell progenitors (CDP) proliferation in the cultures, similar to Flt3L and CDP sorted and cultured in FL10 generate exclusively cDC2. These cDC2 express the transcription factors Irf4, Klf4, and Notch2, and their growth is reduced using BM from Irf4
mice, but the expression of Batf3 and Tcf4 is low. Functionally they respond to TLR3, TLR4, and TLR9 signals by upregulation of the surface maturation markers MHC II, CD80, CD86, and CD40, while they poorly secrete proinflammatory cytokines. Peptide presentation to TCR transgenic OT-II cells induced proliferation and IFN-γ production that was similar to GM-CSF-generated BM-DC and higher than Flt3L-generated DC. Together, our data support that FL10 culture of BM cells selectively promotes CDP-derived ESAM
cDC2 (cDC2B) development and survival in vitro.
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